Subtilases

ABSTRACT

The present invention relates to novel JP170 like subtilases from wild-type bacteria, hybrids thereof and to methods of construction and production of these proteases. Further, the present invention relates to use of the claimed subtilases in detergents, such as a laundry or an automatic dishwashing detergent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/879,156filed on Sep. 10, 2010 (now allowed), which is a divisional of U.S.application Ser. No. 11/575,551 filed on Mar. 19, 2007 (now U.S. Pat.No. 7,816,115) which is a 35 U.S.C. 371 national application ofPCT/DK2005/000597 filed Sep. 21, 2005, which claims priority or thebenefit under 35 U.S.C. 119 of Danish application no. PA 2004 01429filed Sep. 21, 2004 and U.S. provisional application No. 60/611,720filed Sep. 21, 2004, the contents of which are fully incorporated hereinby reference.

SEQUENCE LISTING AND DEPOSITED MICROORGANISMS Sequence Listing

The present invention comprises a sequence listing, which is fullyincorporated herein by reference.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with Deutsche Sammlung von Mikroorganismen andZellkulturen and given the following accession numbers:

Deposit Accession Number Date of Deposit JP170/PD456 hybrid DSM16714 15Sep. 2004 JP170/JT169 hybrid DSM16715 15 Sep. 2004 JT169 DSM16719 15Sep. 2004 PD456 DSM16722 15 Sep. 2004 JP170/JP75 hybrid DSM16712 15 Sep.2004 JP170/JP40 hybrid DSM16713 15 Sep. 2004 JP170/AMRK133 hybridDSM16716 15 Sep. 2004

The deposits contain subtilase genes and hybrid subtilase genes asdescribed in example 1-3.

FIELD OF THE INVENTION

The present invention relates to novel JP170 like subtilases fromwild-type bacteria, hybrids thereof and to methods of construction andproduction of these proteases. Further, the present invention relates touse of the claimed subtilases in detergents, such as a laundry detergentor an automatic dishwashing detergent.

BACKGROUND OF THE INVENTION

Enzymes have been used within the detergent industry as part of washingformulations for more than 30 years. Proteases are from a commercialperspective the most relevant enzyme in such formulations, but otherenzymes including lipases, amylases, cellulases, hemicellulases ormixtures of enzymes are also often used.

The search for proteases with appropriate properties include bothdiscovery of naturally occurring proteases, i.e. so called wild-typeproteases but also alteration of well-known proteases by e.g. geneticmanipulation of the nucleic acid sequence encoding said proteases. Onefamily of proteases, which is often used in detergents, is thesubtilases. This family has been further grouped into 6 differentsub-groups (Siezen R. J. and Leunissen J. A. M., 1997, Protein Science,6, 501-523). One of these sub-groups, the Subtilisin family was furtherdivided into the subgroups of “true subtilisins (I-S1)”, “high alkalineproteases (I-S2)” and “intracellular proteases”. Siezen and Leunissenidentified also some proteases of the subtilisin family, but notbelonging to any of the subgroups. The true subtilisins includeproteases such as subtilisin BPN′ (BASBPN), subtilisin Carlsberg(ALCALASE®, NOVOZYMES A/S) (BLSCAR), mesentericopeptidase (BMSAMP) andsubtilisin DY (BSSDY). The high alkaline proteases include proteasessuch as subtilisin 309 (SAVINASE®, NOVOZYMES A/S) (BLSAVI) subtilisinPB92 (BAALKP), subtilisin BL or BLAP (BLSUBL), subtilisin 147(ESPERASE®, NOVOZYMES A/S), subtilisin Sendai (BSAPRS) and alkalineelastase YaB. Outside this grouping of the subtilisin family a furthersubtilisin subgroup was recently identified on the basis of the 3-Dstructure of its members, the TY145 like subtilisins. The TY145 likesubtilisins include proteases such as TY145 (a subtilase from Bacillussp. TY145, NCIMB 40339 described in WO 92/17577) (BSTY145), subtilisinTA41 (BSTA41), and subtilisin TA39 (BSTA39).

The JP170 subtilase type was first described as protease A in WO88/01293 to Novozymes A/S disclosing four strains producing this type ofprotease. Later U.S. Pat. No. 5,891,701 to Novozymes Biotech disclosedthe amino acid sequence of JP170 and the DNA sequence encoding it. Thepatents JP7-62152 and JP 4197182 to Lion Corp. disclosed the alkalineprotease Yb produced by Bacillus sp. Y that is homologous to JP170 andthe DNA sequence encoding Yb. Bacillus sp. Y also produces the proteaseYa (Geneseq P entry AAR26274). And in addition U.S. Pat. No. 6,376,227to Kao Corp. discloses physical characteristics as well as DNA andpolypeptide sequences of alkaline proteases KP43, KP1790 and KP9860which are also homologous to JP170. Recently genetic engineered variantsof the KP43, KP9860 and Ya proteases among others were disclosed in EP 1209 233, which also disclosed protease A-2 from Bacillus sp. NCIB12513.Kao Corp. also disclosed the proteases KSM-KP9865 and A-1 in US2004/072321. Other known proteases belonging to this group are ProteaseE-1 derived from Bacillus sp. strain No. D6 (FERM P-1592), JP7407101,Protease SD521 derived from Bacillus sp. strain SD-521 (FERM BP-11162),JP9108211, and protease A1 derived from NCIB12289, WO 88/01293 toNovozymes NS.

BRIEF DESCRIPTION OF THE INVENTION

The inventors have isolated novel proteases belonging to the JP170 likeproteases subgroup of the subtilisin family that possess advantageousproperties, such as improved detergent stability.

Furthermore the inventors have inserted truncated forms of the genesencoding various members of this subgroup into the gene encoding theJP170 protease thereby creating hybrid JP170 like proteases exhibitingimproved performance in comparison to the JP170 protease.

The invention therefore in a further embodiment provides hybridproteases.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1, Phylogenetic tree showing the relationship of the maturesubtilase peptide sequences were constructed upon alignment with defaultsettings in the ClustalW function of program MegAlign™ version 5.05 inDNAStar™ program package.

FIG. 2, Matrix with amino acid sequence identities of the enzymes of theinvention and the closest prior art known to the applicant.

DEFINITIONS

Prior to discussing this invention in further detail, the followingterms and conventions will first be defined.

For a detailed description of the nomenclature of amino acids andnucleic acids, we refer to WO 00/71691 page 5, hereby incorporated byreference. A description of the nomenclature of modifications introducedin a polypeptide by genetic manipulation can be found in WO 00/71691page 7-12, hereby incorporated by reference.

The term “subtilases” refer to a sub-group of serine proteases accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases or serine peptidasesis a subgroup of proteases characterised by having a serine in theactive site, which forms a covalent adduct with the substrate. Furtherthe subtilases (and the serine proteases) are characterised by havingtwo active site amino acid residues apart from the serine, namely ahistidine and an aspartic acid residue.

The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisinfamily, the Thermitase family, the Proteinase K family, the Lantibioticpeptidase family, the Kexin family and the Pyrolysin family.

The Subtilisin family (EC 3.4.21.62) may be further divided into 3sub-groups, i.e. I-S1 (“true” subtilisins), I-S2 (highly alkalineproteases) and intracellular subtilisins. Definitions or grouping ofenzymes may vary or change, however, in the context of the presentinvention the above division of subtilases into sub-division orsub-groups shall be understood as those described by Siezen et al.,Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6(1997) 501-523.

The term “parent” is in the context of the present invention to beunderstood as a protein, which is modified to create a protein variant.The parent protein may be a naturally occurring (wild-type) polypeptideor it may be a variant thereof prepared by any suitable means. Forinstance, the parent protein may be a variant of a naturally occurringprotein which has been modified by substitution, chemical modification,deletion or truncation of one or more amino acid residues, or byaddition or insertion of one or more amino acid residues to the aminoacid sequence, of a naturally-occurring polypeptide. Thus the term“parent subtilase” refers to a subtilase which is modified to create asubtilase variant.

The term “hybrid” is in the context of this invention to be understoodas a protein that has been modified by replacing one or more segments ofthe gene encoding the parent protein with corresponding segments derivedfrom genes encoding another protein.

The term “core” in the context of this invention is to be understood asa segment that comprises a substantial part of the subtilase geneincluding the part encoding the active site and a substantial part ofthe rest of the subtilase molecule, to provide unique traits to ahybrid.

The term “modification(s)” or “modified” is in the context of thepresent invention to be understood as to include chemical modificationof a protein as well as genetic manipulation of the DNA encoding aprotein. The modification(s) may be replacement(s) of the amino acidside chain(s), substitution(s), deletion(s) and/or insertions in or atthe amino acid(s) of interest. Thus the term “modified protein”, e.g.“modified subtilase”, is to be understood as a protein which containsmodification(s) compared to a parent protein, e.g. subtilase.

“Homology” or “homologous to” is in the context of the present inventionto be understood in its conventional meaning and the “homology” betweentwo amino acid sequences should be determined by use of the “Similarity”defined by the GAP program from the University of Wisconsin GeneticsComputer Group (UWGCG) package using default settings for alignmentparameters, comparison matrix, gap and gap extension penalties. Defaultvalues for GAP penalties, i.e. GAP creation penalty of 3.0 and GAPextension penalty of 0.1 (Program Manual for the Wisconsin Package,Version 8, August 1994, Genetics Computer Group, 575 Science Drive,Madison, Wis., USA 53711). The method is also described in S. B.Needleman and C. D. Wunsch, Journal of Molecular Biology, 48, 443-445(1970). Identities can be extracted from the same calculation. Thehomology between two amino acid sequences can also be determined by“identity” or “similarity” using the GAP routine of the UWGCG packageversion 9.1 with default setting for alignment parameters, comparisonmatrix, gap and gap extension penalties can also be applied using thefollowing parameters: gap creation penalty=8 and gap extension penalty=8and all other parameters kept at their default values. The output fromthe routine is besides the amino acid alignment the calculation of the“Percent Identity” and the “Similarity” between the two sequences. Thenumbers calculated using UWGCG package version 9.1 is slightly differentfrom the version 8.

The term “position” is in the context of the present invention to beunderstood as the number of an amino acid in a peptide or polypeptidewhen counting from the N-terminal end of said peptide/polypeptide. Theposition numbers used in the present invention refer to differentsubtilases depending on which subgroup the subtilase belongs to.

DETAILED DESCRIPTION OF THE INVENTION Construction of DegeneratedPrimers

Degenerated primers were constructed from an alignment of genes ofalready known proteases such as Ya, KAO KSM-43 and JP170. The primerswere degenerated in order to allow screening for protease gene fragmentsdifferent from Ya, KAO KSM-43 and JP170.

PCR Screening

From the company culture collection a selection of bacterial strainswere included in a PCR screening using the primers SF16A767F andSF16A1802R. The expected size of the PCR product was 1050 nucleotides.All PCR products of the expected size were sequenced in two sequencereaction using one of each of the same two primers. The nucleotidesequences were translated to amino acid sequences, and the diversityanalysed by comparative peptide sequence analysis.

Based on the results of the screening a number of enzymes were selectedfor further investigation. The selected enzymes are shown in FIG. 1, andthey both represent new enzyme molecules and representatives of theprior art. The enzymes selected for further investigation are JP40,JP75, JT169, AMRK133 and PD456, which can be seen as forming a separategroup in FIG. 1. Also hybrid subtilases produced as described below canbe seen in FIG. 1.

Based on these results the inventors decided to move on with a dualapproach; expression of the PCR product by in frame fusions to N and Cterminal parts of the known protease of Bacillus halmapalus strain JP170and inverse PCR to get the full sequences of selected enzymes.

Expression of Hybrid Proteases Description of SOE PCR

By SOE PCR(SOE: Splicing by Overlapping Extension) hybrid gene productscomprising 5 segments were generated as described in Example 2. Thehybrid subtilase genes are used for production of a mature proteaseenzyme of about 433 amino-acids and a molecular weight of approximately45 kd. The first segment is the nucleotide sequence encoding the prosequence of JP170 protease (that is not a part of the mature protease)and 40 amino acids of the N terminal of the mature JP170 protease. Thisis followed by a fusion primer segment encoding 8 amino acids (thissegment may contain sequence variation due to the degeneration of theprimer SF16A767F). The third segment is encoding the approximately 343amino acid long core. This segment includes the sequence encoding theactive site of the protease. This is followed by a fusion primer segmentencoding 7 amino acids (this segment may contain variation due to thedegeneration of the primer SF16A1802R). The fifth segment is encodingthe 35 amino acids of the C terminal of the JP170 protease.

SOE PCR products based on core segments from the following strains weregenerated: JP40 (SEQ ID NO:5), PD456 (SEQ ID NO:7), JP75 (SEQ ID NO:9),JT169 (SEQ ID NO:11), AMRK133 (SEQ ID NO:13) (the SEQ ID number of thegene sequence encoding the mature hybrid protease is indicated inbrackets).

The core of the subtilase of the invention may comprise 50-420 aminoacid residues, preferably 50-100 amino acid residues, 100-150 amino acidresidues, 150-200 amino acid residues 200-250 amino acid residues,250-300 amino acid residues, 300-350 amino acid residues, 350-400 aminoacid residues, 400-420 amino acid residues. Especially preferred is acore segment comprising approximately 343 amino acid residues.

The N terminal end of the core segment is located in one of positions1-10, 10-20, 20-30, 30-40, 40-50, 50-60 or 60-70 of the subtilase of SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. The Cterminal end of the core segment is located in one of positions 70-80,80-90, 90-100, 100-150, 150-200, 200-250, 250-300, 300-320, 320-340,340-360, 360-380, 380-400, 400-420 of the subtilase of SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. In a preferredembodiment the core of the subtilase of the invention comprises theamino acids in position 49-392 of the hybrids JP40 (SEQ ID NO:6), PD456(SEQ ID NO:8), JP75 (SEQ ID NO:10), JT169 (SEQ ID NO:12), AMRK133 (SEQID NO:14).

The core sequence preferably has 94% identity with the amino acids inposition 49-392 of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12or SEQ ID NO:14. More preferably the core sequence has 95% identity, 96%identity, 97% identity, 98% identity or 99% identity with SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.

The corresponding nucleotides encoding the core segment can be seen inany of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ IDNO:13. In a preferred embodiment the core of the subtilase of theinvention is encoded by the nucleotides in position 145-1177 of thehybrids JP40 (SEQ ID NO:5), PD456 (SEQ ID NO:7), JP75 (SEQ ID NO:9),JT169 (SEQ ID NO:11), AMRK133 (SEQ ID NO:13).

The N and C terminals of the hybrids of the present invention couldequally well be selected from other subtilases, such as BLSCAR, BMSAMP,BASBPN or BSSDY of 1-S1, BLSAVI, BAALKP, BLSUBL or subtilisin 147 of1-S2, a members of the TY145 like subtilases, or another member of theJP170 like subtilases.

The lengths of the N and C terminal sequences vary from 1 toapproximately 150 amino acid residues. Preferably the length of theterminals are 1-20 amino acid residues, 20-40 amino acid residues, 40-60amino acid residues, 60-80 amino acid residues, 80-100 amino acidresidues, 100-120 amino acid residues, 120-150 amino acid residues.

The subtilase hybrids of the invention are preferable produced by use ofthe fusion primers described in Example 2, but other suitable primersmay equally well be used.

Cloning of the Hybrid Protease

The PCR fragment was cloned into plasmidpDG268NeoMCS-PramyQ/Prcrylll/crylllAstab/Sav (U.S. Pat. No. 5,955,310)and transformed in Bacillus subtilis. Protease positive colonies wereselected and the coding sequence of the expressed enzyme from theexpression construct was confirmed by DNA sequence analysis.

Cloning and Expression of Full Length Subtilase of the Invention InversePCR

Inverse PCR was performed with specific DNA primers designed tocomplement the DNA sequence of the core PCR product and chromosomal DNAextracted from the appropriate bacterial strain. Inverse PCR was made onthe strains JT169 and PD456. The inverse PCR products were nucleotidesequenced to obtain the region encoding the N and C terminal parts ofthe gene.

Production of Full Length Subtilase

The subtilase genes were amplified with specific primers withrestriction sites in the 5′ end of primers that allow gene fusion withthe Savinase signal peptide of plasmidpDG268NeoMCS-PramyQ/Prcrylll/crylllAstab/Sav (U.S. Pat. No. 5,955,310).Protease positive colonies were selected and the coding sequence of theexpressed enzyme from the expression construct was confirmed by DNAsequence analysis.

Subtilases of the Invention

The subtilase is in the context of the present invention to beunderstood as including the members of the novel subgroup of FIG. 1:JP40, PD456, JP75, JT169, AMRK133 and homologous or hybrids thereof.According to the identity matrix of FIG. 2 the amino acid sequenceidentity of the closest related prior art subtilase is 94.7%.

Thus, the subtilase of the present invention is at least 95% identicalwith SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12 or SEQ ID NO:14. In particular said subtilase may be atleast 96%, at least 97%, at least 98% or at least 99% identical with SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12 or SEQ ID NO:14.

The subtilase of the present invention is encoded by an isolated nucleicacid sequence, which nucleic acid sequence has at least 85% identitywith SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11 or SEQ ID NO:13. Preferably, said nucleic acid sequence hasat least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity with thenucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13.

Further the isolated nucleic acid sequence encoding a subtilase of theinvention hybridizes with a complementary strand of the nucleic acidsequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13 preferably under lowstringency conditions, at least under medium stringency conditions, atleast under medium/high stringency conditions, at least under highstringency conditions, at least under very high stringency conditions.

Hybridization

Suitable experimental conditions for determining hybridization between anucleotide probe and a homologous DNA or RNA sequence involvespresoaking of the filter containing the DNA fragments or RNA tohybridize in 5×SSC (Sodium chloride/Sodium citrate, Sambrook et al.1989) for 10 min, and prehybridization of the filter in a solution of5×SSC, 5×Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989),followed by hybridization in the same solution containing aconcentration of 10 ng/ml of a random-primed (Feinberg, A. P. andVogelstein, B. (1983) Anal. Biochem. 132:6-13), ³²P-dCTP-labeled(specific activity>1×10⁹ cpm/μg) probe for 12 hours at ca. 45° C. Forvarious stringency conditions the filter is then washed twice for 30minutes in 2×SSC, 0.5% SDS and at least 55° C. (low stringency), morepreferably at least 60° C. (medium stringency), still more preferably atleast 65° C. (medium/high stringency), even more preferably at least 70°C. (high stringency), and even more preferably at least 75° C. (veryhigh stringency).

Variants Combined Modifications

The present invention also encompasses any of the above mentionedsubtilase variants in combination with any other modification to theamino acid sequence thereof. Especially combinations with othermodifications known in the art to provide improved properties to theenzyme are envisaged.

Such combinations comprise the positions: 222 (improves oxidationstability), 218 (improves thermal stability), substitutions in theCa²⁺-binding sites stabilizing the enzyme, e.g. position 76, and manyother apparent from the prior art.

In further embodiments a subtilase variant described herein mayadvantageously be combined with one or more modification(s) in any ofthe positions:

27, 36, 56, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 120,123, 159, 167, 170, 206, 218, 222, 224, 232, 235, 236, 245, 248, 252 and274 (BPN' numbering).Specifically, the following BLSAVI, BLSUBL, BSKSMK, and BAALKPmodifications are considered appropriate for combination:K27R, *36D, S56P, N76D, S87N, G97N, S101G, S103A, V104A, V104I, V104N,V104Y, H120D, N123S, G159D, Y167, R170, Q206E, N218S, M222S, M222A,T224S, A232V, K235L, Q236H, Q245R, N248D, N252K and T274A.Furthermore variants comprising any of the modifications S101G+V104N,S87N+S101G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V1041 orN76D+V104A, or other combinations of the modifications K27R, N76D,S101G, S103A, V104N, V104Y, V1041, V104A, N123S, G159D, A232V, Q236H,Q245R, N248D, N252K, T274A in combination with any one or more of themodification(s) mentioned above exhibit improved properties.A particular interesting variant is a variant, which, in addition tomodifications according to the invention, contains the followingsubstitutions:

S101G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K.

Moreover, subtilase variants of the main aspect(s) of the invention arepreferably combined with one or more modification(s) in any of thepositions 129, 131 and 194, preferably as 129K, 131H and 194Pmodifications, and most preferably as P129K, P131H and A194Pmodifications. Any of those modification(s) are expected to provide ahigher expression level of the subtilase variant in the productionthereof.

Methods for Expression and Isolation of Proteins

To express an enzyme of the present invention the above mentioned hostcells transformed or transfected with a vector comprising a nucleic acidsequence encoding an enzyme of the present invention are typicallycultured in a suitable nutrient medium under conditions permitting theproduction of the desired molecules, after which these are recoveredfrom the cells, or the culture broth.

The medium used to culture the host cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). The mediamay be prepared using procedures known in the art (see, e.g., referencesfor bacteria and yeast; Bennett, J. W. and LaSure, L., editors, MoreGene Manipulations in Fungi, Academic Press, CA, 1991).

If the enzymes of the present invention are secreted into the nutrientmedium, they may be recovered directly from the medium. If they are notsecreted, they may be recovered from cell lysates. The enzymes of thepresent invention may be recovered from the culture medium byconventional procedures including separating the host cells from themedium by centrifugation or filtration, precipitating the proteinaceouscomponents of the supernatant or filtrate by means of a salt, e.g.ammonium sulphate, purification by a variety of chromatographicprocedures, e.g. ion exchange chromatography, gelfiltrationchromatography, affinity chromatography, or the like, dependent on theenzyme in question.

The enzymes of the invention may be detected using methods known in theart that are specific for these proteins. These detection methodsinclude use of specific antibodies, formation of a product, ordisappearance of a substrate. For example, an enzyme assay may be usedto determine the activity of the molecule. Procedures for determiningvarious kinds of activity are known in the art.

The enzymes of the present invention may be purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J-C Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

When an expression vector comprising a DNA sequence encoding an enzymeof the present invention is transformed/transfected into a heterologoushost cell it is possible to enable heterologous recombinant productionof the enzyme. An advantage of using a heterologous host cell is that itis possible to make a highly purified enzyme composition, characterizedin being free from homologous impurities, which are often present when aprotein or peptide is expressed in a homologous host cell. In thiscontext homologous impurities mean any impurity (e.g. other polypeptidesthan the enzyme of the invention) which originates from the homologouscell where the enzyme of the invention is originally obtained from.

Detergent Applications

The enzyme of the invention may be added to and thus become a componentof a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations,especially for automatic dish washing (ADW).

In a specific aspect, the invention provides a detergent additivecomprising the enzyme of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas a protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e. pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g. of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Preferred commercially available protease enzymes include Alcalase™,Savinase™, Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S),Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. fromB. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131,253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202. Preferred commercially available lipase enzymes includeLipolase™ and Lipolase Ultra™ (Novozymes A/S).

Amylases: Suitable amylases (α and/or β) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, α-amylases obtained fromBacillus, e.g. a special strain of B. licheniformis, described in moredetail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include Celluzyme™, Renozyme® andCarezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g. from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e. a separate additive or a combined additive, canbe formulated e.g. as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of e.g. the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions any enzyme, in particular the enzyme ofthe invention, may be added in an amount corresponding to 0.01-100 mg ofenzyme protein per litre of wash liquor, preferably 0.05-5 mg of enzymeprotein per litre of wash liquor, in particular 0.1-1 mg of enzymeprotein per litre of wash liquor.

The enzyme of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202 which is herebyincorporated as reference.

Typical powder detergent compositions for automated dishwashing include:1)

Nonionic surfactant 0.4-2.5% Sodium metasilicate 0-20% Sodium disilicate3-20% Sodium triphosphate 20-40% Sodium carbonate 0-20% Sodium perborate2-9% Tetraacetyl ethylene diamine (TAED) 1-4% Sodium sulphate 5-33%Enzymes 0.0001-0.1%2)

Nonionic surfactant (e.g. alcohol ethoxylate)  1-2% Sodium disilicate2-30% Sodium carbonate 10-50%  Sodium phosphonate  0-5% Trisodiumcitrate dehydrate 9-30% Nitrilotrisodium acetate (NTA) 0-20% Sodiumperborate monohydrate 5-10% Tetraacetyl ethylene diamine (TAED)  1-2%Polyacrylate polymer 6-25% (e.g. maleic acid/acrylic acid copolymer)Enzymes 0.0001-0.1%     Perfume 0.1-0.5%  Water 5-103)

Nonionic surfactant 0.5-2.0% Sodium disilicate 25-40% Sodium citrate30-55% Sodium carbonate 0-29% Sodium bicarbonate 0-20% Sodium perboratemonohydrate 0-15% Tetraacetyl ethylene diamine (TAED) 0-6% Maleicacid/acrylic acid copolymer 0-5% Clay 1-3% Polyamino acids 0-20% Sodiumpolyacrylate 0-8% Enzymes 0.0001-0.1%4)

Nonionic surfactant 1-2% Zeolite MAP 15-42%  Sodium disilicate 30-34% Sodium citrate 0-12%  Sodium carbonate 0-20%  Sodium perboratemonohydrate 7-15%  Tetraacetyl ethylene diamine (TAED) 0-3% Polymer 0-4%Maleic acid/acrylic acid copolymer 0-5% Organic phosphonate 0-4% Clay1-2% Enzymes 0.0001-0.1%    Sodium sulphate Balance5)

Nonionic surfactant  1-7% Sodium disilicate 18-30% Trisodium citrate10-24% Sodium carbonate 12-20% Monopersulphate (2 KHSO₅•KHSO₄•K₂SO₄)15-21% Bleach stabilizer  0.1-2% Maleic acid/acrylic acid copolymer 0-6% Diethylene triamine pentaacetate,  0-2.5% pentasodium salt Enzymes0.0001-0.1%   Sodium sulphate, water BalancePowder and liquid dishwashing compositions with cleaning surfactantsystem typically include the following ingredients:6)

Nonionic surfactant 0-1.5% Octadecyl dimethylamine N-oxide dihydrate0-5% 80:20 wt. C18/C16 blend of octadecyl dimethylamine 0-4% N-oxidedihydrate and hexadecyldimethyl amine N- oxide dihydrate 70:30 wt.C18/C16 blend of octadecyl bis 0-5% (hydroxyethyl)amine N-oxideanhydrous and hexadecyl bis (hydroxyethyl)amine N-oxide anhydrousC₁₃-C₁₅ alkyl ethoxysulfate with an average degree of 0-10% ethoxylationof 3 C₁₂-C₁₅ alkyl ethoxysulfate with an average degree of 0-5%ethoxylation of 3 C₁₃-C₁₅ ethoxylated alcohol with an average degree0-5% of ethoxylation of 12 A blend of C₁₂-C₁₅ ethoxylated alcohols withan 0-6.5% average degree of ethoxylation of 9 A blend of C₁₃-C₁₅ethoxylated alcohols with an 0-4% average degree of ethoxylation of 30Sodium disilicate 0-33% Sodium tripolyphosphate 0-46% Sodium citrate0-28% Citric acid 0-29% Sodium carbonate 0-20% Sodium perboratemonohydrate 0-11.5% Tetraacetyl ethylene diamine (TAED) 0-4% Maleicacid/acrylic acid copolymer 0-7.5% Sodium sulphate 0-12.5% Enzymes0.0001-0.1%Non-aqueous liquid automatic dishwashing compositions typically includethe following ingredients:7)

Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0-10.0% Alkalimetal silicate 3.0-15.0% Alkali metal phosphate 20.0-40.0% Liquidcarrier selected from higher glycols, polyglycols, polyoxides,glycolethers 25.0-45.0% Stabilizer (e.g. a partial ester of phosphoricacid and a C₁₆-C₁₈ alkanol) 0.5-7.0% Foam suppressor (e.g. silicone)0-1.5% Enzymes 0.0001-0.1%8)

Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0-10.0% Sodiumsilicate 3.0-15.0% Alkali metal carbonate 7.0-20.0% Sodium citrate 0.0-1.5% Stabilizing system (e.g. mixtures of finely divided  0.5-7.0%silicone and low molecular weight dialkyl polyglycol ethers) Lowmolecule weight polyacrylate polymer 5.0-15.0% Clay gel thickener (e.g.bentonite) 0.0-10.0% Hydroxypropyl cellulose polymer  0.0-0.6% Enzymes0.0001-0.1%  Liquid carrier selected from higher lycols, Balancepolyglycols, polyoxides and glycol ethersThixotropic liquid automatic dishwashing compositions typically includethe following ingredients:9)

C₁₂-C₁₄ fatty acid 0-0.5% Block co-polymer surfactant 1.5-15.0%   Sodiumcitrate  0-12% Sodium tripolyphosphate  0-15% Sodium carbonate  0-8%Aluminium tristearate 0-0.1% Sodium cumene sulphonate 0-1.7%Polyacrylate thickener 1.32-2.5%   Sodium polyacrylate 2.4-6.0%  Boricacid 0-4.0% Sodium formate 0-0.45%  Calcium formate 0-0.2% Sodiumn-decydiphenyl oxide disulphonate 0-4.0% Monoethanol amine (MEA)0-1.86%  Sodium hydroxide (50%) 1.9-9.3%  1,2-Propanediol 0-9.4% Enzymes0.0001-0.1%    Suds suppressor, dye, perfumes, water BalanceLiquid automatic dishwashing compositions typically include thefollowing ingredients:10)

Alcohol ethoxylate 0-20% Fatty acid ester sulphonate 0-30% Sodiumdodecyl sulphate 0-20% Alkyl polyglycoside 0-21% Oleic acid 0-10% Sodiumdisilicate monohydrate 18-33% Sodium citrate dihydrate 18-33% Sodiumstearate 0-2.5% Sodium perborate monohydrate 0-13% Tetraacetyl ethylenediamine (TAED) 0-8% Maleic acid/acrylic acid copolymer 4-8% Enzymes0.0001-0.1%Liquid automatic dishwashing compositions containing protected bleachparticles typically include the following ingredients:11)

Sodium silicate 5-10%  Tetrapotassium pyrophosphate 15-25%  Sodiumtriphosphate 0-2% Potassium carbonate 4-8% Protected bleach particles,e.g. chlorine 5-10%  Polymeric thickener 0.7-1.5%    Potassium hydroxide0-2% Enzymes 0.0001-0.1%    Water Balance12) Automatic dishwashing compositions as described in 1), 2), 3), 4),6) and 10), wherein perborate is replaced by percarbonate.13) Automatic dishwashing compositions as described in 1)-6) whichadditionally contain a manganese catalyst. The manganese catalyst may,e.g., be one of the compounds described in “Efficient manganesecatalysts for low-temperature bleaching”, Nature 369, 1994, pp. 637-639.

Materials and Methods Method for Producing a Subtilase Variant

The present invention provides a method of producing an isolated enzymeaccording to the invention, wherein a suitable host cell, which has beentransformed with a DNA sequence encoding the enzyme, is cultured underconditions permitting the production of the enzyme, and the resultingenzyme is recovered from the culture.

When an expression vector comprising a DNA sequence encoding the enzymeis transformed into a heterologous host cell it is possible to enableheterologous recombinant production of the enzyme of the invention.Thereby it is possible to make a highly purified subtilase composition,characterized in being free from homologous impurities.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed subtilase may conveniently be secreted into the culture mediumand may be recovered there-from by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulfate, followed by chromatographic procedures such asion exchange chromatography, affinity chromatography, or the like.

Example 1 PCR Screening

The core part of protease gene was amplified in a PCR reaction thatincluded 50 U/ml of Ampli-taq™ DNA polymerase (Perkin Elmer) 10×Amplitaq buffer (final concentration of MgCl₂ is 1.5 mM) 0.2 mM of eachof the dNTPs (dATP, dCTP, dTTP and dGTP) 0.2 pmol/μl of the primersSF16A767F (CNATGCATGAAGCNTTCCGCGG, SEQ ID NO:15) (“N” is degenerationintroduced by insertion of inosine)) andSF16A1802R(CNACGTTGTTNCNGCCATCCC, SEQ ID NO:16) and 1 μl template DNA.Template DNA was recovered from the various Bacillus strains usingHighPure™ PCR template preparation kit (Boehringer Mannheim art.1796828) as recommended by the manufacturer for DNA recovery frombacteria. The quality of the isolated template was evaluated by agarosegel electrophoresis. If a high molecular weight band was present thequality was accepted. PCR was run in the following protocol: 94° C., 2minutes 40 cycles of [94° C. for 30 seconds, 52° C. for 30 seconds, 68°C. for 1 minute] completed with 68° C. for 10 minutes. PCR products wereanalysed on a 1% agarose gel in TAE buffer stained with Ethidium bromideto confirm a single band of app. 1050 nucleotides. The PCR product wasrecovered by using Qiagen™ PCR purification kit as recommended by themanufacturer. The nucleotide sequences were determined by sequencing onan ABI PRISM™ DNA sequencer (Perkin Elmer). PCR products of PD456,JT169, JP40, JP75 and AMRK133 were determined. The nucleotide sequenceswere translated to amino acid sequences, and the diversity analysed bycomparative peptide sequence analysis. As can be seen in FIG. 1 thediversity by far exceeded that of the prior art.

Example 2 Production of Subtilase Hybrids Expression of HybridProteases, PCR Amplification

In order to produce an active subtilase based on the nucleotide sequenceinformation of the partial sequencing of Example 1, the core PCR productwas fused to the N and C terminal parts of the JP170 protease gene in aSOE PCR(SOE: Splicing by Overlapping Extension) reaction as describedabove. In the SOE PCR reaction a fusion of three PCR products areproduced. The three PCR products are:

1) The N terminal part of JP170 protease gene. This PCR product isobtained by PCR using the primers PEP192 5′-CCGCGGAATGCTTCATGCATCG-3′(SEQ ID NO:27) and PEP200 5′-GTTCATCGATCTTCTACTATTGGGGCGAAC-3′ (SEQ IDNO:28) and 1 μl template DNA. Template DNA was recovered from thevarious Bacillus strains using HighPure™ PCR template preparation kit(Boehringer Mannheim art. 1796828) as recommended by the manufacturerfor DNA recovery from bacteria. The quality of the isolated template wasevaluated by agarose gel electrophoresis. If a high molecular weightband was present the quality was accepted. PCR was run in the followingprotocol: 94° C., 2 minutes 40 cycles of [94° C. for 30 seconds, 52° C.for 30 seconds, 68° C. for 1 minute] completed with 68° C. for 10minutes. PCR products were analysed on a 1% agarose gel in TAE bufferstained with Ethidium bromide to confirm a single band of app. 700nucleotides.2) The C terminal part of JP170 protease gene. This PCR product isobtained by PCR using the primers

PEP193 5′-GGGATGGCAGAAACAACGTGG-3′ (SEQ ID NO:29) and PEP2015′-TTAAACGCGTTTAATGTACAATCGCTAAAGAAAAG-3′ (SEQ ID NO:30) and 1 μltemplate DNA. Template DNA was recovered from the various Bacillusstrains using HighPure™ PCR template preparation kit (BoehringerMannheim art. 1796828) as recommended by the manufacturer for DNArecovery from bacteria. The quality of the isolated template wasevaluated by agarose gel electrophoresis. If a high molecular weightband was present the quality was accepted. PCR was run in the followingprotocol: 94° C., 2 minutes 40 cycles of [94° C. for 30 seconds, 52° C.for 30 seconds, 68° C. for 1 minute] completed with 68° C. for 10minutes. PCR products were analysed on a 1% agarose gel in TAE bufferstained with Ethidium bromide to confirm a single band of app. 370nucleotides.

3) The core PCR product described in Example 1.

In the SOE PCR reaction the three PCR products are mixed and a fusedproduct is amplified in a standard PCR protocol using the primers PEP200and PEP201 and 1 μl template DNA. Template DNA is a mixture of the threePCR products described above (1-3). These PCR products may be recoveredusing Qiaquick™ spin columns as recommended (Qiagen, Germany). Thequality of the isolated template was evaluated by agarose gelelectrophoresis. PCR was run in the following protocol: 94° C., 2minutes 40 cycles of [94° C. for 30 seconds, 52° C. for 30 seconds, 68°C. for 1 minute] completed with 68° C. for 10 minutes. PCR products wereanalysed on a 1% agarose gel in TAE buffer stained with Ethidium bromideto confirm a single band of app. 1850 nucleotides.

The digested and purified PCR fragment was ligated to the Cla I and MluI digested plasmid pDG268NeoMCS-PramyQ/Prcrylll/crylllAstab/Sav (U.S.Pat. No. 5,955,310).

The ligation mixture was used for transformation into E. coli TOP10F′(Invitrogen BV, The Netherlands) and several colonies were selected forminiprep (QIAprep® spin, QIAGEN GmbH, Germany). The purified plasmidswere checked for insert before transformation into a strain of Bacillussubtilis derived from B. subtilis DN 1885 with disrupted apr, npr andpel genes (Diderichsen et al (1990), J. Bacteriol., 172, 4315-4321). Thedisruption was performed essentially as described in “Bacillus subtilisand other Gram-Positive Bacteria,” American Society for Microbiology, p.618, eds. A. L. Sonenshein, J. A. Hoch and Richard Losick (1993).Transformed cells were plated on 1% skim milk LB-PG agar plates,supplemented with 6 μg/ml chloramphenicol. The plated cells wereincubated over night at 37° C. and protease containing colonies wereidentified by a surrounding clearing zone. Protease positive colonieswere selected and the coding sequence of the expressed enzyme from theexpression construct was confirmed by DNA sequence analysis.

Example 3 Production of Full Length Subtilases Inverse PCR

Three digestions of the chromosomal DNA of the strains PD456 and JT169were made using the restriction enzymes xho1, BamH1 and Pst1. Upondigestion the DNA was separated from the restriction enzymes usingQiaquick™ PCR purification kit (art. 28106, Qiagen, Germany). Thedigestions were religated and subjected to a PCR reaction using primers(PCR primers SEQ ID NO:17-20) designed to recognise the sequence of thePCR product already obtained. The following PCR protocols were applied:94° C. 2 min 30 cycles of [94° C. for 15 s, 52° C. for 30 s, 72° C. for2 min] 72° C. 20 min. Using same PCR amount of primer polymerase andbuffer as above. Alternatively a protocol with 94° C. 2 min 30 cycles of[94° C. for 15 s, 52° C. for 30 s, 68° C. for 3 min] 68° C. 20 min. andreplacing Amplitaq® and Amplitaq® buffer with Long-template TaqPolymerase™ (Boehringer Mannheim) with the buffer supplied with thepolymerease. The PCR reactions were analysed on 0.8% agarose gelsstained with ethidium bromide. All PCR fragments were recovered and thenucleotide sequence was determined by using specific oligo primersdifferent from those used in the PCR reaction (Sequencing primers SEQ IDNO:21-26). In some cases the first primer did not give sufficientnucleotide sequence information to characterise the entire open readingframe of the protease gene. In these cases new primers were appliedeither by using the sequence information obtained with the initialinverse PCR sequencing primer, or by going back to the initial PCRfragment and defining a new primer sequence.

The following primers were used for obtaining the inverse PCR andsequencing:

PCR Primers

PD456 PCR Forward: AGGATTCCCGAACGGAAACCAAGG (SEQ ID NO: 17)PD456 PCR Reverse: TCCGTTTCCAAGTACAGACCCGG (SEQ ID NO: 18)JT169 PCR Forward: TCTCTCTAGTATGGTCTGATGCTCC (SEQ ID NO: 19)JT169 PCR Reverse: TCCGTTTCCAAGAACAGATCCGGC (SEQ ID NO: 20)

Sequencing Primers

JT169 Forward Sequencing ATCTGCTTCTATAACACTGG (SEQ ID NO: 21)JT169 Reverse Sequencing1 TGCGTTCCGTGACCATTTGG (SEQ ID NO: 22)JT169 Reverse Sequencing2 TTTGCGTCAAGTGCCACAGC (SEQ ID NO: 23)JT169 Reverse Sequencing3 TCAACAATTTCATCTATGCC (SEQ ID NO: 24)PD456 Forward Sequencing GCCGTGTAACAATGGACAAG (SEQ ID NO: 25)PD456 Reverse Sequencing1 TCCCTTTATTGGTTGCTCCG (SEQ ID NO: 26)

The gene sequences encoding the mature part of the protease gene ofstrains JT169 and PD456 are shown in SEQ ID NO:1 and SEQ ID NO:3respectively.

Production of the Full Length Subtilase

To produce the subtilases of strain JT169 and PD456 the protease genewas amplified from chromosomal DNA of the wild type strains or from theclones deposited as DSM16719 and DSM16722 using the primers:

(SEQ ID NO: 31) JT169 Expression ForwardAGTTCATCGATCGGGGGAGCTAGCAGCTTCGA (SEQ ID NO: 32)JT169 Expression Reverse TGATTAACGCGTTTAGTTCACAATCGCCAATG(SEQ ID NO: 33) PD456 Expression ForwardAGTTCATCGATCGGGGGGGCTAGCAACTTTGA (SEQ ID NO: 34)PD456 Expression Reverse TGATTAACGCGTTTAGTTCACAATCGCCAATG

These PCR products were digested with restriction enzymes Cla1 and Mlu1(except AA351 that was digested with (Nar1 and Mlu1) and ligated intopDG268neo, and expressed as described in Example 2.

Example 4 Purification and Characterisation Purification

This procedure relates to purification of a 2 liter scale fermentationfor the production of the subtilases of the invention in a Bacillus hostcell. Approximately 1.6 liters of fermentation broth are centrifuged at5000 rpm for 35 minutes in 1 liter beakers. The supernatants areadjusted to pH 6.5 using 10% acetic acid and filtered on Seitz Supra®S100 filter plates.

The filtrates are concentrated to approximately 400 ml using an Amicon®CH₂A UF unit equipped with an Amicon® S1Y10 UF cartridge. The UFconcentrate is centrifuged and filtered prior to absorption at roomtemperature on a Bacitracin affinity column at pH 7. The protease iseluted from the Bacitracin column at room temperature using 25%2-propanol and 1 M sodium chloride in a buffer solution with 0.01dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chlorideadjusted to pH 7.

The fractions with protease activity from the Bacitracin purificationstep are combined and applied to a 750 ml Sephadex® G25 column (5 cmdia.) equilibrated with a buffer containing 0.01 dimethylglutaric acid,0.2 M boric acid and 0.002 m calcium chloride adjusted to pH 6.5.

Fractions with proteolytic activity from the Sephadex® G25 column arecombined and applied to a 150 ml CM Sepharose® CL 6B cation exchangecolumn (5 cm dia.) equilibrated with a buffer containing 0.01 Mdimethylglutaric acid, 0.2 M boric acid, and 0.002 M calcium chlorideadjusted to pH 6.5.

The protease is eluted using a linear gradient of 0-0.1 M sodiumchloride in 2 litres of the same buffer.

In a final purification step subtilase containing fractions from the CMSepharose® column are combined and concentrated in an Amicon®ultrafiltration cell equipped with a GR81PP membrane (from the DanishSugar Factories Inc.).

Example 5 Stability of Subtilases

The stability of the produced subtilases was evaluated in a standardWestern European dishwashing tablet detergent without other enzymes thanthe experimentally added subtilases. The stability of the subtilases isdetermined as the residual proteolytic activity after incubation of thesubtilase in a detergent.

The formulation of a standard Western European Tablet detergent isdefined as

Component Percentage Non ionic surfactants 0-10% Foam regulators 1-10%Bleach (per-carbonate or per-borate) 5-15% Bleach activators (e.g. TAED) 1-5% Builders (e.g. carbonate, phosphate, tri-phosphate, Zeolite)50-75%  Polymers 0-15% Perfume, dye etc.  <1% Water and fillers (e.g.sodium sulphate) Balance

Assay for Proteolytic Activity

The proteolytic activity was determined with casein as substrate. OneCasein Protease Unit (CPU) is defined as the amount of proteaseliberating about 1 μM of primary amino groups (determined by comparisonwith a serine standard) per minute under standard conditions, i.e.,incubation for about 30 minutes at about 25° C. at pH 9.5.

The proteolytic activity may also be determined by measuring thespecific hydrolysis of succinyl-Ala-Ala-Pro-Leu-p-nitroanilide by saidprotease. The substrate is initially dissolved in for example, DMSO(Dimethyl Sulfoxide) and then diluted about 50 fold in about 0.035 Mborate buffer, about pH 9.45. All protease samples may be diluted about5-10 fold by the same borate buffer. Equal volumes of the substratesolution and sample are mixed in a well of an ELISA reader plate andread at about 405 nm at 25° C. All sample activities and concentrationsare normalized to the standard protease solution activity andconcentration, respectively.

A typical Western European tablet detergent for automated dishwashingwas dissolved (5.5 g/L) in 9° dH water at ambient temperature maximum 30minutes prior to start of analyses. Samples of subtilases were dilutedto a concentration of 2-4 CPU/ml in Britten Robinson buffer (BrittenRobinson buffer is: 40 mM Phosphate, 40 mM Acetate and 40 mM Borate)pH9.5. For the analyses every sample was divided and tested under twoconditions: For the control the subtilase was diluted 1:9 in BrittenRobinson buffer pH9.5 to a final volume of 1 ml. This sample wasanalysed immediately after dilution. For the detergent stability thesubtilase sample was diluted 1:9 in detergent solution (detergentconcentration in the stability test is 5 g/L) these samples wereincubated at 55° C. for 30 minutes prior to analysis by addition ofcasein substrate.

The assay was started by addition of 2 volumes of casein substrate(casein substrate was 2 g of casein (Merck, Hammerstein grade) in 100 mlof Britten Robinson buffer pH 9.5, pH was re-adjusted to 9.5 when thecasein is in solution). Samples are kept isothermic at 25° C. for 30minutes. The reaction was stopped by addition of 5 ml TCA solution (TCAsolution is 89.46 g of Tri-chloric acid, 149.48 g of Sodiumacetate-tri-hydrate and 94.5 ml of glacial acetic acid in 2.5 L ofdeionised water). The samples are incubated at ambient temperature forat least 20 minutes and filtered through Whatman® paper filter no. 42.

400 μl of filtrate is mixed with 3 ml OPA reagent (OPA reagent iscomposed of: 3.812 g of borax, 0.08% EtOH, 0.2% DTT and 80 mg ofo-phthal-dialdehyd in 100 ml water). Absorption at 340 nM is measuredand CPU is calculated from the concentration of free amines on astandard of a solution of 0.01% L-serine (Merck art. 7769).

Enzymatic proteolysis of reference proteases in the typical WesternEuropean tablet detergent:

CPU/L Protease Control Detergent % activity Alcalase 250 31 13% Esperase220 116 53% Savinase 538 21 4% Everlase16L 2383 86 4% Ovozyme 2848 44 2%BLAP-S 36 1 3% JP170 754 370 49%

Enzymatic proteolysis of cloned full length proteases of the inventionin the typical Western European tablet detergent:

CPU/L Clone Control Detergent % activity PD456-1 87 95 109% PD456-2 8491 108% PD456-3 85 93 109% PD456-4 77 84 109% JT169-1 74 65 88% JT169-271 60 85% JT169-3 62 59 95% JT169-4 59 58 98%

Enzymatic proteolysis of cloned hybrid proteases of the invention in thetypical Western European tablet detergent. The reference is JP170:

CPU/I Hybrid Control Detergent % activity JP170 67 36 53% JP170 66 3857% JP75-1 70 78 112% JP75-2 72 82 114% JP40-1 67 79 118% JP40-2 66 78118% PD456-1 74 80 108% PD456-2 74 80 108% JT169-1 83 79 95% JT169-2 8482 97%

As can be seen from the results the subtilases and subtilase hybrids ofthe invention exhibit a greatly improved proteolytic activity afterincubation in a detergent as compared to the prior art subtilase JP170.Therefore, the subtilases and subtilase hybrids of the inventionexhibits improved stability in a detergent as compared to the prior art.

1-21. (canceled)
 22. An isolated subtilase, comprising an amino acidsequence which is at least 95% identical to the sequence of SEQ ID NO:2,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14, and which hasproteolytic activity.
 23. The subtilase of claim 22, comprising an aminoacid sequence which is at least 96% identical to the sequence of SEQ IDNO:2, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
 24. Thesubtilase of claim 22, comprising an amino acid sequence which is atleast 97% identical to the sequence of SEQ ID NO:2, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12 or SEQ ID NO:14.
 25. The subtilase of claim 22,comprising an amino acid sequence which is at least 98% identical to thesequence of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQID NO:14.
 26. The subtilase of claim 22, comprising an amino acidsequence which is at least 99% identical to the sequence of SEQ ID NO:2,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
 27. Thesubtilase of claim 22, which comprises the sequence of SEQ ID NO:2, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
 28. The subtilaseof claim 22, which consists of the sequence of SEQ ID NO:2, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
 29. A detergent compositioncomprising the subtilase of claim 22 and a surfactant.
 30. A nucleicacid sequence encoding the subtilase of claim
 22. 31. The nucleic acidsequence of claim 29 as shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11 or SEQ ID NO:13.
 32. A nucleic acid construct comprisingthe nucleic acid sequence of claim 30 operably liked to one or morecontrol sequences capable of directing the expression of the polypeptidein a suitable expression host.
 33. A recombinant expression vectorcomprising the nucleic acid construct of claim 32, a promoter, andtranscriptional and translation stop signals.
 34. The vector of claim33, further comprising a selectable marker.
 35. A recombinant host cellcomprising the nucleic acid construct of claim
 32. 36. A method forproducing a subtilase, comprising a) cultivating a recombinant host cellof claim 35; and b) recovering the subtilase.
 37. A core subtilase whichhas an which has an amino acid sequence which is 96% identical to thesequence of amino acids from position 49 through position 391 of SEQ IDNO:2, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
 38. Ahybrid subtilase comprising the core sequence of claim 37.